Extended Data Fig. 4: Further analysis of the PhPINK1 oligomeric state.

a, Model built into the PhPINK1(D357A) dodecamer cryo-EM density as in Fig. 2b. b, Side view of the oligomer showing how the N-helix–CTR area facilitates contacts to connect four dimers. c, We were concerned that PhPINK1 oligomerization may only happen with a specific mutant protein and tested whether the oligomer would also form with unphosphorylated WT PhPINK1. WT PhPINK1 is phosphorylated at multiple sites when purified from E. coli (see¹⁰), and was hence dephosphorylated by λ-PP and repurified by SEC as indicated (see Methods). Like the inactive D357A mutant, a prominent oligomer, as well as a dimer/monomer equilibrium is apparent on SEC. Alternatively, the WT protein was first dephosphorylated by λ-PP, then rephosphorylated by adding Mg²⁺/ATP for 1 min, and the kinase and λ-PP were inactivated by EDTA (see Methods). Phosphorylation destabilized the oligomer to a predominant monomeric species, explaining why WT PhPINK1 does not form the oligomer, and suggesting that autophosphorylation resolves the oligomer and dimer (see below). These experiments were part of the purification process and were performed three times with identical results. d, SEC–MALS analysis of TcPINK1 variants, to show that corresponding TcPINK1 constructs (amino acids 117–570) do not show oligomeric behaviour. TcPINK1 purifications revealing monomeric behaviour were performed at least twice, and a SEC–MALS experiment was performed once. e, Residues mediating hydrogen bonds (dotted lines) in the oligomer interfaces. Lack of conservation of oligomer contacts between PhPINK1, TcPINK1 and HsPINK1 likely explain why TcPINK1 does not form an oligomer, and why we do not expect an identical oligomer in HsPINK1.